Characterization of a FourU RNA Thermometer in the 5′ Untranslated Region of Autolysin Gene blyA in the Bacillus subtilis 168 Prophage SPβ

RNA thermometers are noncoding RNA structures located in the 5′ untranslated regions (UTRs) of genes that regulate gene expression through temperature-dependent conformational changes. The fourU class of RNA thermometers contains a specific motif in which four consecutive uracil nucleotides are predicted to base pair with the Shine-Dalgarno (SD) sequence in a stem. We employed a bioinformatic search to discover a fourU RNA thermometer in the 5′-UTR of the blyA gene of the Bacillus subtilis phage SPβc2, a bacteriophage that infects B. subtilis 168. blyA encodes an autolysin enzyme, N-acetylmuramoyl-l-alanine amidase, which is involved in the lytic life cycle of the SPβ prophage. We have biochemically validated the predicted RNA thermometer in the 5′-UTR of the blyA gene. Our study suggests that RNA thermometers may play an underappreciated yet critical role in the lytic life cycle of bacteriophages.

R NA thermometers are mostly cis-acting RNA structural elements found in the 5′ untranslated region (5′-UTR) of a gene. 1,2Typically, RNA thermometers regulate gene expression within a physiologically relevant temperature range of 25−42 °C.In most characterized examples, they result in an increased level of gene expression in response to higher temperatures.This temperature-dependent upregulation occurs as the RNA structure becomes denatured and reveals a previously sequestered gene expression platform. 1,2ourU thermometers make up a well-characterized class of RNA thermometers containing a specific motif in which the ribosome-binding site, the SD sequence, is predicted to base pair in a stem across from four consecutive uracil nucleotides. 3ith an increase in temperature, base pairing within the fourU motif is disrupted, causing destabilization of the stem and increased accessibility to the SD sequence, enabling translation of a downstream gene. 3Several fourU RNA thermometers have been found in the 5′-UTR of genes involved in the heat shock response or bacterial virulence.These include the ompA gene in Yersinia pseudotuberculosis 4 and Shigella dysenteriae, 5 the agsA gene in Salmonella enterica, 3 the toxT gene in Vibrio cholerae, 6 the shuA gene in S. dysenteriae, 7 and the recently discovered glycerol permease fourU RNA thermometer (glpT) in Bacillus subtilis. 8B. subtilis also has a second glycerol permease RNA thermometer (glpF) that is a non-fourU RNA thermometer. 8he homology among several previously characterized fourU RNA thermometer motifs was exploited to search for novel fourU RNA thermometers.The RNA motif search tool, RNArobo, 9 was used to identify potential fourU RNA thermometers based on the predicted secondary structure of the fourU agsA RNA thermometer 3 (Methods in the Supporting Information).The results of our in silico approach revealed a potential RNA thermometer in the 5′-UTR of the blyA gene (also known as yomC), which encodes an autolysin enzyme (N-acetylmuramoyl-L-alanine amidase) that facilitates the hydrolysis of cell wall glycopeptides 10 (Figure 1A,B).This gene is found in the genomes of the B. subtilis phage SPβc2 (SPβ) and its host, the Gram-positive bacterium B. subtilis 168 (B.subtilis).Previously, only one RNA thermometer has been described in a phage genome. 22he prophage SPβ contains the open reading frame for blyA, which plays a crucial role in the lytic activity of the phage in the host cell. 10SPβ integrates into the genome of its bacterial host and has been found to be ubiquitous in B. subtilis 168. 11,12Pβ is a lysogenic phage with a temperature-dependent lytic life cycle in B. subtilis 168. 10,13,14Previous studies have demonstrated that the level of expression of blyA in B. subtilis 168 increases at high temperatures, and its heat-induced expression is necessary for the lysis of the host cell. 10Although it is known that heat stress induces the expression of blyA and the proliferation of the SPβ prophage in B. subtilis 168, 10 the underlying molecular mechanism that drives this process has yet to be elucidated.
RNA thermometer function is dependent upon a conformational change in the RNA secondary structure.RNAfold 15 was employed to predict the full-length structure of the RNA thermometer sequence in the 5′-UTR of blyA (Figure 1C).The predicted secondary structure of the 5′-UTR of blyA is closely related to the predicted structure of the agsA RNA thermometer 3 and contains two hairpins with the fourU motif in the P2 stem.To determine the thermoregulatory function, the 5′-UTR of blyA (5UTR_blyA) was cloned into a reporter plasmid containing an arabinose-inducible pBAD promoter upstream of a heat-stable β-galactosidase (bgaB) from Bacillus stearothermophilus. 16The 5UTR_blyA−bgaB fusion was constructed by replacing the native 5′-UTR of bgaB with the 5′-UTR of blyA (Figure S1).
Temperature-dependent expression was determined by heat induction of Escherichia coli cells expressing the 5UTR_blyA− bgaB fusion.Cells containing 5UTR_blyA−bgaB fusions were incubated for 30 min at 25, 37, or 42 °C.Subsequently, βgalactosidase activity was measured for each temperature (Methods in the Supporting Information).Heat shock of cells expressing 5UTR_blyA-bgaB fusions resulted in heat induction factors [activity in Miller units (M.U.) at 37 °C/25 or 42 °C/ 25 °C] of ∼4.0and ∼5.2-fold at 37 and 42 °C, respectively (Figure 1D).bgaB fusions containing the established agsA RNA thermometer were used as positive controls and demonstrated a similar heat induction profile.As a negative control, the 5′-UTR of a DNA gyrase gene (gyrA), which is not expected to be thermally regulated, was tested and exhibited minimal heat induction of 0.94-and 0.95-fold at 37 and 42 °C, respectively (Figure 1D).These findings demonstrate that the 5′-UTR of blyA modulates reporter gene activity in a temperature-dependent manner, with a notable increase in the level of gene expression at increased temperatures.
To investigate differences between transcriptional and translational control in the system, transcript levels of 5UTR_blyA−bgaB fusions were measured by quantitative real-time PCR (qRT-PCR).Cells were harvested under the same conditions of β-galactosidase assays at 25 and 42 °C.Heat induction resulted in an approximately 2.6-fold increase in transcript abundance at 42 °C, indicating some regulation at the transcriptional level (Figure 1E).Similar increases in transcript levels have been reported for other RNA thermometers, suggesting an additional layer of regulation. 17,18t is currently unknown how transcription of the 5′-UTR of blyA is regulated.
To further validate that the blyA RNA thermometer sequence is directly responsible for the thermal regulation of gene expression, mutations were made to strengthen and stabilize the base pairing of the fourU stem motif.As previously demonstrated with the agsA RNA thermometer, 3 stabilization of the fourU motif results in a decrease in the level of heat induction.The predicted secondary structure of the blyA RNA thermometer was used to design stabilizing mutations to the fourU motif to strengthen the base pairing of P2 (Figure 2A), and β-galactosidase assays of mutants were performed at 25 and 42 °C to determine activity.U41C and U42C mutations in the fourU region across from the SD sequence change G•U wobble base pairs to stronger canonical G-C base pairs.Strikingly, double mutant UU4142CC completely abolished heat induction activity.The U41C stabilizing mutation by itself did not show any decrease in the level of heat induction, while the mutants U42C and CUU394041AAA notably reduced the level of heat induction to ∼1.5-fold (Figure 2B).In addition, thermal analysis comparison of the wild-type 5′-UTR blyA sequence and the double mutant UU4142CC showed that the double mutant's overall melting temperature (75.9 ± 0.4 °C) is considerably higher than that of the wild type (69.4 ± 0.4 °C) (Figure 2C).Altogether, our results indicate that the thermoregulation of the blyA RNA thermometer is directly dependent on the thermal stability of the fourU motif of P2.
The prevailing mechanism through which most RNA thermometers act requires the destabilization of the RNA secondary structure at high temperatures. 19At increased temperatures, the stem containing the RBS undergoes a zipper-like, unwinding melting mechanism, allowing access to the RBS. 2,20To further investigate the conformational changes induced by heat, structural probing, using 2-methylnicotinic acid imidazolide (NAI) in DMSO, was performed on the blyA RNA thermometer sequence.Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) provides secondary structure information at a single-nucleotide resolution.The reactivity of each RNA base is correlated with the flexibility of the 2′-OH, with single-stranded or flexible regions exhibiting increased reactivity in opposition to regions engaged in base pairing or other interactions. 21In vitro SHAPE analysis at 25 and 42 °C reveals a prominent temperature-dependent increase in the reactivity of the fourU motif, indicating that the fourU motif is more flexible and accessible in response to an increase in temperature (Figure 3).The most pronounced increases in reactivity were observed in nucleotides U41 and U42, which were shown to be necessary for heat induction (Figure 2B).Interestingly, despite U41 appearing to have the most increased activity at 42 °C, the U41C mutation does not appear to affect translation (Figure 2B).The U41C single mutation is likely not sufficient to stabilize the fourU motif and influence the overall melting temperature of the structure.Overall, structural probing analysis supports a temperaturedependent change in the flexibility and accessibility of the fourU motif of P2, suggesting an increased level of access for ribosome binding at higher temperatures.
Lastly, to verify if the P2 stem can achieve thermoregulation by itself, a truncated 5UTR_blyA−bgaB fusion containing only the P2 stem (mini-5UTR_blyA) (Figure 4A) was tested.Heat induction of mini-5UTR_blyA−bgaB fusions resulted in a heat induction factors of ∼3.0and ∼3.2-fold at 37 and 42 °C, respectively (Figure 4B).Heat induction is notably reduced when P1 is deleted, although P2 is capable of functioning as a thermometer to a lesser extent.In comparison, a similar fusion containing only the P2 hairpin of the agsA fourU thermometer retains overall heat induction. 3Complete thermoregulation by the blyA thermometer is dependent on the P1 and P2 hairpins to accomplish maximal heat induction.
Although the fourU motif is a common structure found in many RNA thermometers, all previously annotated fourU thermometers have been discovered in bacteria.Additionally, to date, only one other RNA thermometer has been found in a bacteriophage.However, this previously discovered bacteriophage RNA thermometer inhibits gene expression at increased temperatures. 22The field of synthetic biology has also shown more interest in RNA thermometers for their potential use in biotechnology and medicine.In this study, we discovered a fourU RNA thermometer located in the 5′-UTR of the blyA gene within the genomes of the SPβ prophage and its host, B. subtilis 168.−12 Furthermore, it has been reported that increasing the temperature of the host to 50 °C for a brief period leads to the activation of SPβ through heat induction, causing the phage to replicate and the host cell to undergo lysis.−13 Our results revealed that the 5′-UTR of the blyA gene is an RNA thermometer, indicating an additional level of thermoregulation in SPβ.Our study suggests that similar RNA thermometers may regulate bacteriophage lytic life cycles and may be more common than previously anticipated.

Figure 1 .
Figure 1.Discovery of an RNA thermometer upstream of blyA.Genome loci of (A) B. subtilis SPβ bacteriophage and (B) B. subtilis 168 showing the RNA thermometer upstream of the gene blyA that encodes an N-acetylmuramoyl-L-alanine amidase.(C) Secondary structure prediction of the 5′-UTR of blyA.The fourU U41 and U42 residues and start codon are underlined, and the Shine-Dalgarno (SD) sequence is boxed.(D) Heat induction factor of bgaB fusions with the blyA 5′-UTR.Expression at 25, 37, and 42 °C was compared to a positive control, the agsA fourU RNA thermometer, and a negative control, DNA gyrase (gyrA) (mean ± standard deviation; n = 3 biological replicates).(E) Relative transcript levels of the 5′-UTR of blyA at 25 and 42 °C measured by qRT-PCR.The transcript levels of the 5′-UTR of blyA were normalized to reference gene gyrA (mean ± standard deviation; n = 3 biological replicates, each with 3 technical replicates).

Figure 2 .
Figure 2. Characterization of the blyA 5′-UTR.(A) Secondary structure prediction of the 5′-UTR of blyA, showing mutated nucleotides.The start codon is underlined, and the Shine-Dalgarno (SD) sequence is boxed.(B) Heat induction factor of bgaB fusions with mutations of the blyA 5′-UTR.Expression at 25 and 42 °C of mutants was compared to that of negative control DNA gyrase (gyrA) (mean ± standard deviation; n = 3 biological replicates).(C) Circular dichroism thermal analysis of the wild-type 5′-UTR of blyA (orange) and mutant variant UU4142CC (green) recorded at 290 nm.

Figure 3 .
Figure 3. Structural probing analysis of the 5′-UTR of blyA.(A) SHAPE reactivity plots for the 5′-UTR of blyA at 25 °C (blue) and 42 °C (red) (mean ± standard deviation; n = 3 technical replicates).U41 and U42 are underlined.(B) Secondary structure predictions of the 5′-UTR of blyA with SHAPE reactivity values at 25 and 42 °C.Nucleotides are color-coded on the basis of the intensity of SHAPE reactivity.U41 and U42 are underlined.